Exosome Inhibition of Hematopoietic Colonies

Human cord blood was obtained from the Institute of Transfusion Medicine, University of Pittsburgh, and mononuclear cells (MNCs) were isolated using Ficoll-gradient centrifugation. Exosomes isolated from AML patients’ plasma or leukemia cell lines (1–50 μg protein) were used for the colony forming cell assays. Exosomes were suspended in PBS and plated in 35 mm culture dishes. MNCs were combined with methylcellulose media containing recombinant cytokines (SCF, 50 ng/ml; IL-3, 10 ng/ml; GM-CSF,10 ng/ml; and erythropoietin, 1 U/ml) for use with human cells (Stem Cell Technologies, Cambridge, Boston, MA, USA) as previously described [24 (link)]. Cells were plated in triplicate wells at the concentration of 5 × 10⁴ per mL of media. The cell mixture was placed on top of the exosomes. Plates were incubated at 37 °C in 5% CO₂ for 12–14 d before colonies were enumerated using an inverted microscope. CFU-GM and CFU-GEMM colonies were differentiated and reported along with total colony counts. For reversal of inhibition experiments, exosomes were combined with either control media or Diprotin A (5 mM), a DPP4 inhibitor, for 4 h prior to plating [24 (link), 26 (link)].